Some of the numerous feeding strategies are more appropriate than others for certain types of cell culture production systems. Once a nutrient supplement has been identified as described in Part 1 of this three-part review (1), a supplementation strategy must be chosen. Supplementing at too great a rate may expose log-phase cells to stresses such as increased osmolality and lactate levels that would inhibit biomass expansion. But inadequate supplementation can lead to early apoptosis through rapid depletion of selected important components. For commercially available supplements, guidelines often suggest specific protocols that will usually yield good results and can be further optimized by subsequent experimentation.

Additional culture-specific methods are available to laboratories with access to monitoring equipment. As mentioned in Part 1, monitoring one component is an excellent way of approaching relative nutrient homeostasis in the bioreactor, especially with stoichiometrically balanced nutrient supplements (1). They may be added semicontinuously (e.g., daily or twice daily), with each addition providing enough supplement to cover its expected consumption until the next addition. Supplements also can be added continually to match predicted consumption, but this approach is more generally applicable at the research scale than in commercial production.

PRODUCT FOCUS: ALL PROTEINS

PROCESS FOCUS: PRODUCTION

WHO SHOULD READ: MANUFACTURING AND PROCESS DEVELOPMENT

KEYWORDS: CULTURE MEDIA, FED-BATCH, PERFUSION, MEDIA SUPPLEMENTS, PROCESS CONTROL, AND DESIGN OF EXPERIMENTS

LEVEL: INTERMEDIATE

Supplementation of cell cultures generally refers to adding nutrients that replace those consumed, but another type of supplement can be considered. Cell metabolic pathways are important to optimizing protein expression because of the dichotomous pathway of biomass expansion and product synthesis. Cells that are expanding rapidly may not have the highest specific productivity (output per cell), but once maximum cell density is reached, specific productivity can peak and be held at high levels for extended periods of time. Nutrient supplements can be designed to promote both phases. So nutritional supplements (those containing major nutrients such as glucose and specific amino acids that are consumed during the expansion phase) represent one type of supplement. And metabolic supplements (those containing molecules to guide cells toward maximum productivity) represent the other type. Timing their addition may promote superior productivity.

A number of molecules have been identified as helpful in the production phase of cell culture. When glucose is replaced by a more slowly metabolized hexose such as galactose or mannose, a slower metabolism results with correspondingly lowered lactic acid production. At the same time, inducers of protein synthesis such as butyrate and pentanoic acid can be used for switching cell machinery from further replication at the plateau phase into protein expression. Numerous small molecules are being researched as protein synthesis stimulators in high-throughput systems.

Once parameters are optimized, nutrient supplementation can usually double cell productivity, at least. Here, different nutrient supplements and feeding strategies developed by Invitrogen scientists improve cell performance (Figure 1) (Click to enlarge)

Figure 1: (Click to enlarge)

Nutrient Supplementation of Bioreactors

Bioreactor supplementation choices are made based on the timing and quantity of supplement to add to a culture. If a balanced, clone-specific nutrient supplement is used, one measurable component can determine the choice. With a low-glucose set point, Fike et al. (2) used computer-controlled peristaltic pumping of a stoichiometrically balanced supplement in a continuous fed-batch process to maintain a homogeneous nutrient environment that yielded a fourfold productivity increase.

Another relatively common method of determining when to add supplement is by monitoring OUR, the oxygen uptake rate (3). Tracking OUR to maintain nutrient levels provides an aerobic culture metabolism for maximum energy efficiency while minimizing lactate and ammonia production (3). This approach works whether supplementing just glucose and/or glutamine or a more complex supplement. Supplement is added based on oxygen consumption to control glucose and glutamine at low levels without depletion or overabundance. OUR-based supplementation can increase cell densities and improve productivity. With proper control, metabolism shifting toward increased lactate production during the growth phase may be counterbalanced by lactate consumption during the production phase (4).

Other methods of supplementation involve monitoring parameters such as pH, lactic acid, glutamine, dissolved oxygen (DO) levels, or glucose (3). Even turbidity can be monitored for supplementation purposes. Another approach is to add supplement proportional to the amount of base addition used to control pH. As a culture expands in cell number and consumes nutrients, it usually produces lactate, which reduces pH (kept in line by base addition and thus the cue for adding more supplement).

In another example of fed-batch process control, Sauer et al. used off-line measurement of glucose to guide supplementation of a concentrated protein-free supplement that was scalable from 15 L to 750 L, with a 7.6-fold improvement of monoclonal antibody (MAb) yield (5). Their approach was to seek a rapid, generic, fed-batch process using a commercially available “low-content” base medium and feeding the culture with a “rich” supplement. Somewhat analogous to this, Xie et al. specifically designed a base medium to reduce lactate and ammonia, then supplemented their culture with nutrients stoichiometrically shown to be depleting, allowing supplementation only as needed (6). Nutrients were kept from becoming depleted, lactate and ammonia levels were significantly lower, and cell density and productivity were increased by fivefold and 10-fold respectively.

Spens et al. (7) used a dual approach to derive a supplement that boosted productivity >11-fold. First, they performed depletion studies on bioreactor cultures to identify those macronutrients that could be quantitated easily. Nonmeasurable compounds were then identified by separate shake cultures of various supplement categories in a design of experiments (DoE) study. Combining data yielded a superior supplement that included lipids as an important feed component.

Fractional factorial design is another useful approach when it comes to assessing micronutrients for supplementation — e.g., trace elements, growth factors, and insulin — that may not be readily quantitated or that can interact with other supplement component(s). Sandadi et al. showed identification and quantitative modification of important components with interdependencies within a supplement (8).

Although most current supplementation regimens are of the nutrient-only type, biphasic approaches may become popular if they can enhance productivity. Success would require some knowledge of cell growth kinetics. Throughout the exponential phase, only nutrient supplements should be used to stimulate maximal cell mass expansion. Only as a culture has entered the plateau phase (but before a significant drop-off in viability) should a metabolic supplement such as butyrate be used.

Timing for addition of a metabolic inducer type of supplement (to immediately inhibit cell growth in favor of improved protein production) may need additional clarity. Sitton et al. (9) showed with CHO culture that monitoring gross viability or total cell count is not reliable for identifying the end of expansion and beginning of the culture's stationary production phase. The team noted significant variation despite control of bulk culture parameters. They found that a nonviable subpopulation of cells (identified by automated flow cytometry) precisely identified the end of exponential proliferation, signaling the start of production feeding. The result was significantly improved total bioreactor cell count available for the production phase.

One other aspect of supplementation involves the potential need to replace nonconsumed components. In fed-batch cultures, up to 40–50% supplementation may be optimal. Nonconsumed chemicals thus may end up at half the concentration of that in the original medium, potentially dropping them to suboptimal levels. It may be advisable to add back some important components to yield a 1× “original” concentration. This may be especially important for vitamins, trace elements, and certain salts such as phosphates that have been shown to enhance cell product synthesis (10).

Supplementation Rates: A disadvantage of perfusion methods is product dilution resulting in high purification volumes. One solution may be combining multiple rounds of perfusion rate reduction with a more concentrated basal perfusion medium (11). Greater nutrient concentrations in the medium should allow perfusion rates to be lowered. Presumably this could provide much better productivities than fed-batch culture because some wastes are reduced even with a lowered perfusion rate. The challenge would be to choose a rate yielding the highest product concentration that also keeps toxic molecules at relatively low levels. Another limitation would be finite nutrient solubility in the basal medium — and the potential for adding grossly elevated levels of nutrients early in such a culture.

Slow feeding of concentrates with a fed-batch process based on known nutrient requirements usually improves productivity. Product synthetic pathways, however, may require minimal levels of sensitive or critical components that could easily be left out or added at too low a rate if consumption rates are unknown, jeopardizing product formation. Senger et al. compared a fixed, rapid feeding rate with a metabolically determined one and showed improved productivity using the former, pointing out that it may be better to err on the side of higher supplementation rates to cover unquantified cell needs (12). Using similar reasoning, Takagi et al. supplemented basal medium with fivefold amino acids and vitamins to increase volumetric yield of tissue plasminogen activator (tPA) by 3.6-fold, which the team considered to be critical to an economically viable production process (13).

In addition to stable production platforms, transient gene expression (TGE) is also benefited by nutrient supplementation (14,15). Sun et al. improved HEK 293 production of green fluorescent protein (GFP) and erythropoeitin (EPO) by supplementing with a 5× amino-acid concentrate based on DMEM/F12 in a fed-batch culture (15). Yet another approach was taken by Dempsey et al. using GS NSO cells adapted to grow in a serum-free medium without glutamine. The team used several rounds of culture supplementation followed by analytical spent-medium analysis to sequentially develop a nutrient supplement. In the final version, it showed depletion of no basic components and resulted in a final 10-fold improvement of product expression (16).

In an attempt to standardize methods of culture nutrient supplementation, Zeng et al. studied numerous stoichiometric ratios and related various cell culture parameters together (e.g., ammonium yield from glutamine or lactate from glucose) to determine which were most significant to cell productivity (17). They also presented an equation specifying oxygen consumption. The observed ratios were relatively constant and cell-line independent, making it possible to use them in controlling difficult-to-determine components and develop relatively fast feeding strategies for a number of cell lines and media. The team found that amino acid metabolism is a major consumer of oxygen and that using an OUR/glucose ratio is an excellent way to monitor and control culture supplementation rates.

Although nutrient supplementation is often thought to be important mainly as a culture reaches plateau and available nutrients are lowered or depleted, evidence suggests that considering a day-0 feed may prove beneficial. Fassnacht et al. showed that supplementation at the start of a culture led to decreased lactate and increased membrane stability during the death phase and increased MAb production. Addition at the beginning of the culture promoted beneficial metabolic pathways from initiation (18). Although components for a day-0 feed could be added to the basal medium itself for the same effect, adding them separately kept the basal medium “unadulterated” for use in a range of different clone culture options.